Development of an on-line SPR-digestion-nanoLC-MS/MS system for the quantification and identification of interferon-gamma in plasma.

ABSTRACT

An automated, on-line system for protein quantification and identification, employing Surface Plasmon Resonance (SPR), enzymatic protein digestion, nanoLC and tandem-MS (MS/MS), has been developed. For the experiments recombinant human interferon-gamma (rhIFN-gamma) in buffer or diluted bovine plasma was used as a model protein. Upon injecting 90muL of a 1mugmL(-1) solution of rhIFN-gamma in diluted plasma at a flow rate of 10muLmin(-1), 320fmol of protein was reproducibly bound to the sensor surface. After desorption of the isolated protein from the SPR surface using 10mM glycine pH 1.3, on-line digestion, nanoLC and MS/MS analysis, rhIFN-gamma could be identified on basis of peptide masses and MS/MS fragmentation data. A sequence recovery of 66% was found when a pepsin micro reactor was used. For a trypsin micro reactor the sequence recovery was 50%. In the latter case, the desorbed protein solution was pH-tuned with a TRIS buffer for optimal enzyme activity. With the identified trypsin- and pepsin-produced peptides and because parts of their amino acid sequences overlap, the protein sequence can be largely elucidated showing the potential for the analysis of unknown proteins. The SPR-digestion-nanoLC-MS/MS platform provides unattended analysis of a sample within 60min.